The overall goal of this project is to investigate the feasibility of using peptide analogues derived from glutamic acid decarboxylase (GAD), a diabetogenic autoantigen, for the treatment of insulin-dependent diabetes mellitus (IDDM) in non-obese diabetic (NOD)/HLA-DR4 transgenic mice. Susceptibility to diabetes is linked to the MHC class II locus in both NOD mice and humans, and 95% of Caucasians with IDDM express either HLA-DR3 and/or -DR4. The disease is believed to be mediated, in part, by autoantigen-sensitized MHC class II restricted T cells that home to the pancreatic islets and release inflammatory cytokines. A possible role for GAD in IDDM is suggested by the presence of GAD specific T cell responses and antibodies in prediabetic humans and mice, and by the observation that IDDM can be prevented in mice by the induction of GAD-specific tolerance. Recent studies indicate that altered peptide ligands (APLs) that share sequence homology with native antigenic determinants but have amino acid substitutions in selected TCR contact residues can influence the differentiation of naive CD4+ T cells into Th1 or Th2 effectors and alter the cytokine profile of antigen specific clones. In this project APLs that down-regulate GAD-specific Th1 responses and/or induce anti- inflammatory cytokines (IL-4, IL-10, TGF-beta) will be designed and used as the basis for an immunotherapeutic approach to IDDM. These studies will utilize a recently developed algorithm for predicting the immunogenicity of HLA-DR4 binding peptides, in combination with NOD/DR4 transgenic mice and a novel in vitro system for eliciting GAD-specific human T cell responses. The specific aims are to 1) define the immunodominant HLA-DR4 restricted T cell determinants of GAD and generate mouse and human Th1 CD4+ T cell clones to these determinants; 2) demonstrate that these clones home to pancreatic islets and accelerate diabetes when administered to NOD/DR4 mice; 3) generate APLs that either anergize or alter the cytokine profile of the GAD-specific Th1 clones, or directly induce anti-inflammatory cytokines, in vitro; and 4) demonstrate that the APLs postpone the onset or reduce the severity of diabetes in NOD/DR4 mice, and investigate the mechanisms responsible for the observed effects. If these studies are successful, the results should provide the basis for clinical trials in patients with IDDM.